The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. While CM constitute a short-lived product of intestinal fat absorption, VLDL and, particularly, LDL are responsible for cholesterol transport into tissues, including for example, the arterial walls. In contrast, HDL are directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as "reverse cholesterol transport" (RCT).
The "protective" role of HDL has been confirmed in a number of studies (e.g. Miller et al. Lancet, 1977:965-968 and Whayne et aL Atherosclerosis 1981;39:411-419). In these, the elevated levels of LDL, less so of VLDL, seem to be clearly associated with an increased cardiovascular risk, whereas high HDL levels seem to confer cardiovascular protection. The protective role of HDL has been further strongly supported by the in vivo studies, showing that HDL infusions into rabbits may hinder the development of cholesterol induced arterial. lesions (Badimon et al, Lab. Invest. 60, 455-61, 1989)) and/or induce regression of same (Badimon et al, J Clin Invest. 85, 1234-41, 1990).
Recent interest in the study of the protective mechanism/s of HDL has been focussed on apolipoprotein Al (Apo Al), the major component of HDL. High plasma levels of Apo Al are associated with reduced risk of CHD and presence of coronary lesions (Maciejko et al,. N Engl J Med 1983;309:385-389, Sedlis et al,. Circulation 1986;73:978-984).
Plasma Apo Al is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al, Biochem Biophys Res Commun 1978;80:623-630). Apo Al is synthesized as a 267 amino acid precursor in the cell. This pre-pro-apoliprotein is processed by N-terminal cleavage first intracellularly where 18 amino acids are lost and then with a further cleavage of 6 amino acids in the plasma or the lymph by the activity of specific proteases.
The major structural requirement of the Apo Al molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al, FEBS Lett 1974;38:247-253). This structure allows for the main biological activities of Apo Al, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation.
Another recently described property of Apo Al is its antiviral activity. This has been reported from in vitro studies and is exerted both against Herpes virus strains (Srinivas R V et al,, Virology, 1756, 48-57, 1990) and also against the Human Immunodeficiency virus, HIV, (Owe et al,., J Clin Invest, 86, 1142-50, 1990). This activity seems to be exerted by way of an interaction between amphipatic helical portions of Apo Al and envelope glycoproteins of the viruses.
In vitro studies indicate that complexes of Apo Al and lecithin can promote the efflux of free cholesterol from cultured arterial smooth muscle cells (Stein. et al,. Ciochem Biophys Acta 1975;380:106-118). By this mechanism HDL can also reduce the proliferation of these cells (Yoshida et al, Exp Mol Pathol 1 984;41 :258-266).
More recently, the infusion of Apo Al or of HDL in experimental animals has been shown to exert significant biochemical changes, as well as to reduce the extent and severity of atherosclerotic lesions. After an initial report by Maciejko and Mao (Arteriosclerosis 1982;2:407a), Badimon et al, (see the two quoted studies above) found that they could significantly reduce the extent of atherosclerotic lesions (-45%) and their cholesterol ester content (-58,5%) in cholesterol-fed rabbits, by infusing HDL (d=1.063-1.325 g/ml). They also found that the infusions of HDL led to a close to a 50% regression of established lesions. It was able to be shown also (Esper et al. Arteriosclerosis 1987;7:523a) that infusions of HDL can markedly change the plasma lipoprotein composition of Watanabe rabbits with inherited hypercholesterolemia, which develop early arterial lesions. In these, HDL infusions can more than double the ratio between the protective HDL and the atherogenic LDL.
The potential of HDL to prevent arterial disease in animal models has been further stimulated by the observation that Apo Al can exert a fibrinolytic activity in vitro (Saku et al, Thromb Res 1985;39:1-8). Ronneberger (Xth Int Congr Pharmacol, Sidney 1987, p 990) demonstrated that extractive Apo Al can increase fibrinolysis in beagle dogs and in Cynomologous monkeys. A similar activity can be noted in vitro on human plasma. This author was able to confirm a reduction of lipid deposition and arterial plaque formation in Apo Al treated animals.
The apolipoprotein Al-Milano (Apo Al-M) is the first described molecular variant of human Apo Al (Franceschini et al, J Clin Invest 1980;66:892-900). It is characterized by the substitution of Arg 173 with Cys (Weisgraber et al, J Biol Chem 1983;258:2508-2513). The mutant apoprotein is transmifted as an autosomal dominant trait and 8 generations of carriers have been identified (Gualandri et al, Am J Hum Genet 1984;37:1083-1097).
The status of the Apo Al-M carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease; indeed, by examination of the genealogic tree it appears that these subjects may be "protected" from atherosclerosis.
The mechanism of the possible protective effect of Apo Al-M in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Francheschini et al,. J Biol Chem 1985;260:1632-1635). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal Al. Moreover, apolipoprotein/lipid complexes are more susceptible to denaturation, thus suggesting that lipid delivery is also improved in the case of the mutant.
The therapeutic use of the apolipoprotein Apo Al-M mutant is presently limited by the lack of a method allowing the preparation of said apolipoproteins in sufficient amount and in a suitable form.
Another very specific feature of the Apo Al-M, is its capacity to form dimers with itself and complexes with Apo All, in both cases because of the presence of the Cys residue. From studies of blood fractions containing a mixture of Apolipoproteins, there were indications, showing that the presence of dimers and complexes in the circulation may be responsible for the increased elimination half-life of these in the carriers, recently described in clinical studies (Gregg et al,. NATO ARW on Human Apolipoprotein Mutants: From Gene Structure to Phenotypic Expression, Limone SG, 1988).
Apo Al-M dimers (Apo Al-M/Apo Al-M) act as an inhibiting factor in the interconversion of HDL particles in vitro (Franceschini et al, J Biol Chem 1990;265:12224-12231).
Earlier studies of mixtures containing the dimer have been based on Apo Al-M separated from natural blood from persons with Apo Al-M, which has thus only been obtainable in small quantities.
The difficulty of producing Apo Al and particularly Apo Al-M from plasma fractionation is quite considerable (Franceschini et al, J Biol Chem 1985;260:16321-16325). The isolation and production cannot be done on a big scale, as only a small amount of the raw material is available. Furthermore, there are several risks associated with plasma fractionation products, such as contamination with infectious agents. It is essential that this is avoided.
Attempts have been made to produce human Apo Al, by way of the recombinant DNA technology. In the European patent publication No. 0267703 the preparation of Apo Al from E.coli is described. The process describes a chimeric polypeptide where the Apo Al moiety is fused to the N-terminal amino acid residues of beta-galactosidase or to one or more IgG-binding domains of Protein A, or to the pro sequence of human Apo Al.
The expression of Apo Al and Apo Al-M in yeast strains and the use of the produced components in the treatment of atherosclerosis and cardiovascular diseases is disclosed in WO90/12879. The genes encoding the Apo Al and Apo Al-M were provided with DNA-sequences encoding a yeast-recognizable secretion and processing signals fused upstream to the gene for the mature proteins. A modified MF-alpha-1-leader sequence was used in which the last residues were: HisGlySerLeuAspLysArg.